Feline infectious peritonitis virus vaccines

ABSTRACT

The isolation, identification and in vitro propagation of feline infectious peritonitis virus and preparation of vaccines for immunization of animals of the genus Felis, family felidae therefrom are disclosed.

This invention relates to virus isolation and preparation of vaccines.In particular, the invention relates to the isolation and identificationof feline infectious peritonitis (FIP) virus, to the in vitropropagation of FIP virus in tissue culture, to preparation of live virusvaccines containing FIP virus alone or in combination with other felineviruses and to processes for preparing and using such vaccines.

Feline infectious peritonitis (FIP) is a disease of both domestic andwild cats characterized by progressive debilitation and, in the "wet" oreffusive form, a fibrinous peritoneal exudate. The virus affects most ofthe internal organs of the animal and, in the acute phase of thedisease, is invariably fatal with a mortality rate of nearly 100%(Gaskin in "Current Veterinary Therapy VI. Small Animal Practice", R. W.Kirk, ed., W. B. Saunders Co. (1977), pgs. 1305-1308). Cases of FIPdisease have been reported throughout the world. The virus itself ishighly contagious, affecting kittens as well as adult cats of all ages.

The FIP virus has recently been identified as a Coronavirus by Horzinekand Osterhaus, Arch. Virol. 59:1 (1979), and is, thus, the firstidentified feline Coronavirus. Attempts to culture the FIP virus invitro have, however, been unrewarding. Growth of the virus in cellcultures of the peritoneal exudate of infected kittens was reported byPedersen, Am. J. Vet. Res. 37:567 (1976), but attempts to grow the FIPagent in primary and continuous cell line cultures were unsuccessful.Hoshino and Scott, Cornell Veterinarian 68:411 (1978), likewise reportedunsuccessful attempts to isolate FIP virus in vitro. Those researchersalso described the replication of FIP virus in organ cultures of felinesmall intestine. Propagation of FIP virus in suckling mouse brain wasachieved by Osterhaus et al., Zentralbl. Veterinarmed. 25B(4):301(1978). The mouse-adapted virus strain has been replicated in the brainsof suckling rats and hamsters by Horzinek and Osterhaus, supra.

Although attempts have been made to protect cats against FIP disease byadministration of inactivated crude tissue vaccines, all such attemptshave failed to protect the animals (Gaskin, supra). In addition,virulent FIP virus, administered as a homogenized liver suspension, wasused to vaccinate swine in a cross-protection study between FIP virusand porcine transmissible gastroenteritis (TGE) virus carried out byWoods and Pedersen, Veterinary Microbiology, 4:11 (1979), withinconclusive results.

One aspect of the present invention consists of safe and effectivevaccines for the protection of animals belonging to the genus Felis,family felidae against FIP disease. A monovalent vaccine prepared fromlive FIP virus is preferably administered by the oral, intranasal orintraocular vaccination routes. Such vaccine preferably contains fromabout 10² to about 10⁴ TCID₅₀ /ml of the FIP virus with suitablecarriers and/or stabilizers. Combination polyvalent vaccines containingvaccinal amounts of modified live feline viruses such as felinerhinotracheitis virus, calicivirus and/or panleukopenia virus and theFIP virus described herein are also objects of this invention and may beadministered via oral, intranasal or intraocular routes. Preferably, anysuch vaccine will contain from about 10² to about 10⁴ TCID₅₀ /ml of theFIP virus and suitable carriers and/or stabilizers.

Yet another aspect of the present invention is the FIP virus, isolatedin plaque-purified form and adapted to grow in cell cultures asdescribed herein.

The FIP virus used to prepare the vaccines of this invention is isolatedfrom the organs or tissues of animals infected with the virus. Theorgans or tissues are disaggregated into single cells or may be used asorgan cultures. Growth of cells from the diseased tissue occurs withevidence of FIP virus infection, such as formation of multinucleatedgiant cells and/or cytopathic changes known to be typical of aCoronavirus. Supernatant fluids from such in vitro cultures containvirus which is infectious for susceptible cats when administered by theoral, nasopharyngeal, intramuscular, subcutaneous, intraperitoneal orintravenous routes.

The FIP virus grows readily in feline cells from any source, for examplespleen, mesenteric lymph node or endothelial cells. The virus replicatesin cells obtained directly from cat tissues and in continuous cell linesobtained originally from the feline. The virus will replicate at atemperature of about 30° C. to about 40° C., preferably from 34°-37° C.,in cells growing in a multitude of growth media, such as Eagles' BME orMEM, McCoy's or Hanks' balanced salt solution plus lactalbumin, and isnot affected by the type of growth medium used. Replication can beachieved in the presence of as much as 15% fetal calf serum which mayalso contain penicillin, streptomycin, fungazone or equivalentantibacterial or antifungal agents.

The FIP virus can also be propagated using co-cultivation techniqueswith cells from normal animals or feline cell lines. The cells of theinfected tissue and the normal cells are mixed together at varyingconcentrations, preferably 50%-50%, or any proportions so desired aslong as the amount of diseased cells will exceed 1.0% of the final cellpreparation. The infectious virus is transferred from diseased cells tonormal cells as demonstrated by formation of multinucleated cells and/orcytopathology. Infectious virus can be demonstrated in the supernatantfluid of such cultures by subsequent infection of susceptible animals orby any in vitro test system, such as routine viral titrations in cellcultures, serum neutralization tests, fluorescent antibody tests, enzymelabeled immunoabsorbent assays or other recognized in vitro methods.

After ascertaining that the virus has multiplied in the normal cellcultures, the cell cultures can be frozen and thawed one or more timesand the virus in the supernatant fluid transferred to other normal cellcultures. This method can be repeated indefinitely with cultivation atfrom about 30° C. to about 40° C., preferably at 34°-37° C. For example,the FIP virus isolated and propagated in accordance with this inventionhas been serially passed on stable cell lines at least 80 times withoutexhibiting any changes. Virus content of the supernatant fluid increaseswith each serial cell passage until a maximum virus concentration isreached. Measurement of the virus content of the cell culture can beascertained by in vitro tests as mentioned above or by infectingsusceptible cats by any of the routes previously mentioned.

The FIP virus isolated and propagated in accordance with this inventionwas deposited with the American Type Culture Collection in Rockville,Maryland on Aug. 21, 1979 and has been assigned accession numberVR-2004. It will be freely available on request upon issuance of thisapplication, or any foreign equivalent thereof, as a patent.

DETAILED DESCRIPTION OF THE INVENTION Isolation and Propagation of theFIP Virus

The FIP virus used to make the vaccines of the present invention wasoriginally isolated by cultivation of cells from a disaggragated spleenaseptically removed from an FIP infected cat which had succumbed to theinfection. To determine whether the virus was infectious for susceptibleanimals, mascerated tissue suspension and extratissue fluid wasadministered by intraperitoneal injection to a SPF (specific pathogenfree) cat which subsequently died of typical FIP symptoms.

To propagate the FIP virus, the removed spleen was minced into piecesapproximately 4-5 mm square, washed three times with Hanks' balancedsalt solution and placed in a 1000 ml trypsinizing flask. Approximately200 ml of 0.25% trypsin prepared in Hanks' balanced salt solution wasadded. A teflon magnet was added to the trypsinization flask and theentire flask was placed on a magnetic stirrer. The tissue fragments werestirred for 15 minutes, after which time the tissue fragments wereallowed to settle and the supernatant trypsin was decanted anddiscarded. Another 200 ml of trypsin was added to the flask and thetissue fragments were again stirred for one hour. The cell containingsupernatant fluid was then decanted under aseptic conditions and placedat 4° C. Trypsin solution was added and the procedure was repeated untilall cells were removed from the tissue fragments and only organ stromaremained (about four times).

The trypsin-cell suspension was filtered through sterile gauze and thensedimented at 600 rpm approximately 600×gravity in a refrigeratedLourdes centrifuge. The resultant cell pack was washed with Hanks'balanced salt solution and resedimented. This procedure was repeatedthree times to eliminate traces of residual trypsin.

The packed cells were then diluted to 1 to 100 with growth mediumconsisting of McCoy's medium supplemented with 10% fetal calf serum and100 units of penicillin, 500 mcg of streptomycin and 10 units offungazone per ml. One hundred (100) ml of cell suspension was placedinto a 1000 ml plastic Corning bottle and 5 ml into 60 mm Petri platescontaining 5 mm×16 mm glass cover slips. The Corning bottle wasincubated at 37° C. in a regular incubator and the Petri plates at 37°C. in a humidified carbon dioxide incubator.

Within 24 hours, cells attached to the surface of the bottle and Petriplates. Multiplication of the cells was allowed to proceed until amonolayer of cells covered the exposed surfaces under the medium.Versene and trypsin were then added to the bottle and the cells weredetached from the bottle surface, resuspended in 200 ml of growth mediumand replanted into two Corning plastic bottles and 60 mm Petri platescontaining glass cover slips. The cell monolayer which had formed on theglass cover slips was stained with hematoxlyn and eosin histostains andobserved on a Leitz microscope.

Multinucleated cells containing as many as 10-20 nuclei were noted inthe stained cell preparations. Production of such multinucleated orgiant cells is evidence of virus infection (probably formed by fusion ofinfected cells) and is a characteristic of all well establishedCoronaviridae.

The second passage of infected spleen cells produced abundantmultinucleated cells on the glass slides which could be observed inunstained living cell preparations. Supernatant fluid was removed fromthe infected cells in the plastic bottle and, when introduced orallyinto susceptible SPF cats from the Norden Laboratories' cat colony,produced typical FIP disease symptoms and death.

A total of six serial culture passages was carried out with the FIPinfected cells. Disease symptoms characteristic of FIP were seen in allSPF cats administered the supernatant fluid from each serial passage ofthe infected cell culture.

FIP virus was also isolated by co-cultivation of a serially propagablecell line with cells obtained from infected feline lymph nodes accordingto the following procedure:

An SPF cat from the Norden Laboratories cat colony was orally infectedwith virus obtained from the supernatant fluid of FIP infected catspleen cells. When the infected cat was in extremis from infection andexhibited typical FIP symptoms, the animal was euthanitized and themesenteric lymph nodes were aseptically removed.

The mesenteric lymph nodes were cut into pieces approximately 1 mm insize, washed three times with Hanks' balanced salt solution and thenplaced in a 1000 ml trypsinizing flask with 200 ml of 0.25% trypsin. Ateflon covered magnet was introduced into the flask and rotation of themagnet was allowed to occur over a magnetic stirrer. Disaggregation ofthe cells was allowed for one hour. The fluid and cells were decantedfrom the tissue fragments and stored at 4° C. Fresh trypsin (200 ml) wasplaced on the tissue fragments. This procedure was continued until onlytissue stroma remained and nearly all cells were suspended insupernatant fluid (about three times).

The trypsin-cell suspension was then sedimented at 600 rpm for 10minutes in a refrigerated Lourdes centrifuge. The resultant cell packwas washed with Hanks' balanced salt solution and resedimented in a likemanner. This procedure was repeated three times to remove residualtraces of trypsin.

The cells thus obtained consisted of numerous cell types such ashematopoietic cells, epithelial cells and some unidentified cell types.These cells were suspended in Eagle's MEM (minimum essential medium)plus 10% embryonic calf serum and regular tissue culture antibioticsconsisting of penicillin, streptomycin and fungizone. The lymph nodecells were then mixed with a feline continuous cell line (NL-FK-1) sothat enough NL-FL-1 cells were present to form a monolayer of cells in a1000 ml plastic Corning screw cap bottle. The combined cells wereallowed to incubate at 37° C. until a monolayer of cells could bemicroscopically observed. A 60 mm Petri plate containing glass coverslips was inoculated with 5 ml of the dual cell suspension and incubatedin a humidified incubator in the presence of 5% carbon dioxide and 95%air.

When a monolayer of cells was observed (within 48 hours followinginfection), the glass cover slips were removed and stained withhematoxlyn-eosin. Cytopathology indicating Coronaviridae infection wasobserved, which is characterized by giant multinucleated cell formation,cell degeneration with retraction and detachment from the glass surface.

Supernatant fluid from the plastic bottle was removed and introducedinto normal or uninfected NL-FK-1 cells. These cells were incubated at37° C. until multinucleation of cells could be observed in an unstainedor living state.

Serial cultivation of the FIP virus in the NL-FK-1 cell line was carriedout. Supernatant fluid from virus passages 1 to 30 caused typical FIPclinical symptoms and death when introduced orally, intraperitoneally,intravenously, subcutaneously or intramuscularly into susceptible cats.The virus also caused a cytopathic change in the NL-FK-1 cell lineresulting in destruction of the cell monolayer. The virus can beserially passed continually and indefinitely in the NL-FK-1 cell line.

Co-cultivation of FIP infected cells from other feline tissues orcultures can also be carried out.

In order to conclusively establish and identify the FIP virus as aCoronavirus, supernatant fluids from cell cultures were sedimented at25,000×gravity for two hours, then the resultant pellet of virus wassedimented through a sucrose gradient, subjected to phosphotungstic acidstaining and examined in a transmission electron microscope. Theaggregate virus particles demonstrated the typical morphologiccharacteristics of a Coronavirus, for example infectious bronchitisvirus of chickens, the prototype of the Coronaviridae. The FIP virusthus isolated was deposited in the American Type Culture Collection onAug. 21, 1979 and given accession number VR-2004.

Preparation and Use of FIP Virus Vaccines

A live virus vaccine was prepared by diluting virulent FIP virussuspensions 1 to 100. A dose of 1.0 ml of the diluted virus suspensionwas administered to two susceptible cats via the oral route. Three weekslater a 1 to 50 dilution of the same material was administered to thesame cats in identical manner. No disease symptoms were observed ineither of the treated animals.

Subsequent dilutions of the FIP virus were made, increasing the virusconcentration until each cat received 1.0 ml of a 1 to 5 dilution. Noclinical symptoms of FIP were observed after the administration of eachsuch virus dilution. Three weeks after administration of the finaldilution, the cats were challenged with 1.0 ml of undiluted FIP infectedtissue suspension by the oral route. Two susceptible animals weresimilarly infected at the same time. The unvaccinated animals exhibitedclassic FIP disease symptoms and died at seven and ten days postinfection. The vaccinated animals remained healthy and free of anysymptoms of disease.

After three weeks, the immunity of the two surviving vaccinated animalswas again challenged. Two control unvaccinated cats succumbed to thedisease, while the immunized animals remained healthy and free ofdisease symptoms. A third challenge three weeks after the secondchallenge and a forth challenge six months after the third resulted inthe vaccinated cats remaining healthy and free of disease symptoms andall control unvaccinated animals (two per challenge) succumbing to FIPdisease.

A further aspect of this invention is the preparation and use ofcombination vaccines consisting of vaccinal amounts of the FIP virus andone or more known feline viruses. For example, live virus felinevaccines consisting of vaccinal amounts of modified felinerhinotracheitis virus, calicivirus, and/or panleukopenia virus combinedwith FIP virus can be prepared for oral, intraocular or intranasaladministration. Such polyvalent vaccine will, preferably, contain fromabout 10% to about 20 % total volume of FIP virus. An example of apolyvalent vaccine contemplated by this invention consists of from10-20% of FIP virus, 20-30% of feline rhinotracheitis virus, 20-30% ofcalicivirus and 10-20% of panleukopenia virus (all percentages based ontotal volume).

The preparation and use of such combination vaccines is carried outaccording to procedures described herein or within the knowledge ofthose skilled in the art of vaccine production and use.

What is claimed is:
 1. A live feline infectious peritonitis virusvaccine for oral, intranasal or intraocular administration capable ofinducing immunity in animals of the genus Felis, family felidae withoutserious side effects comprising a vaccinal amount of from about 10² toabout 10⁴ TCID₅₀ /ml of live feline infectious peritonitis virus which,in the undiluted state, can be demonstrated to be infectious forsusceptible cats by any known in vitro method or by causing typicalfeline infectious peritonitis clinical disease symptoms and death whenadministered by the oral, nasopharyngeal, intramuscular, subcutaneous,intraperitoneal or intravenous routes and which can be serially passedon stable cell lines without exhibiting any change, and a carriertherefor.
 2. The feline infectious peritonitis virus vaccine of claim 1wherein the feline infectious peritonitis virus is isolated andpropagated in cell cultures at about 30°-40° C.±2° C.
 3. The felineinfectious peritonitis virus vaccine of claim 2 wherein the felineinfectious peritonitis virus is isolated and propagated in feline cells.4. The feline infectious peritonitis virus vaccine of claim 3 whereinthe feline infectious peritonitis virus is isolated and propagated inprimary feline cells.
 5. The feline infectious peritonitis virus vaccineof claim 4 wherein the feline infectious peritonitis virus is isolatedand propagated in feline kidney cells.
 6. The feline infectiousperitonitis virus vaccine of claim 3 wherein the feline infectiousperitonitis virus is passaged at least once in feline cells.
 7. Thefeline infectious peritonitis virus vaccine of claim 5 wherein thefeline infectious peritonitis virus is passaged at least once in felinekidney cells.
 8. The feline infectious peritonitis virus vaccine ofclaim 7 wherein the feline infectious peritonitis virus is passaged from1 to about 100 times in feline kidney cells.
 9. The feline infectiousperitonitis virus vaccine of claim 8 wherein the feline infectiousperitonitis virus is passaged from 1 to about 30 times in feline kidneycells.
 10. The feline infectious peritonitis virus vaccine of claim 1,2, 3, 4, 5, 6, 7, 8 or 9 wherein the feline infectious peritonitis virusis ATCC No. VR-2004.
 11. A method of vaccinating animals of the genusFelis, family felidae comprising administering to said animals by theoral, intranasal or intraocular route the live feline infectiousperitonitis virus vaccine of claim 1 or
 10. 12. A combination live virusfeline vaccine for oral, intranasal or intraocular administrationcapable of inducing immunity in animals of the genus Felis, familyfelidae without serious side effects comprising a vaccinal amount offrom about 10² to about 10⁴ TCID₅₀ /ml of live feline infectiousperitonitis virus and vaccinal amounts of one or more vaccinal felineviruses selected from modified feline viral rhinotracheitis virus,calicivirus and panleukopenia virus, and a carrier therefor.
 13. Thecombination vaccine of claim 12 which contains from about 10% to about20% total volume of feline infectious peritonitis virus.
 14. Thecombination vaccine of claim 13 which contains from 10-20% total volumeof feline infectious peritonitis virus, 20-30% total volume of modifiedfeline rhinotracheitis virus, 20-30% total volume of calicivirus and10-20% total volume of panleukopenia virus.
 15. A method of vaccinatinganimals of the genus Felis, family felidae comprising administering tosaid animals by the oral, intranasal or intraocular route a combinationvaccine of claim 12, 13 or
 14. 16. The combination live virus felinevaccine of claim 12 wherein the feline infectious peritonitis virus isATCC No. VR-2004.
 17. A method of vaccinating animals of the genusFelis, family felidae comprising administering to said animals by theoral, intranasal or intraocular route the combination vaccine of claim16.